1. Field of the Invention
The present invention relates to a method for improving the detection of morphine and its analogues in mammalian body fluids, particularly in equine body fluids, and especially in equine urine and serum protein-free filtrate, utilizing .beta.-glucuronidase from Patella vulgata for glucuronide hydrolysis.
2. Background Art
Many drugs, including the narcotic analgesics, are metabolized by conjugation with glucuronic acid (Glucuronic Acid, Free and Combined, ed. G. J. Dutton, Academic Press, New York, N.Y., 1966, 88-116, 301-357 and 457-488). In humans, 80-95% of morphine recovered from urine is in the conjugated form [S. Y. Yeh, J. Pharmacol. Exp. Ther. 192, 201-210 (1975); E. L. Way et al, Pharmacol. Rev. 12, 383-446 (1960)]. The conjugated form is not detected in most assays; however, if the analytical procedure includes hydrolysis, the concentration of detectable morphine in urine can be enhanced. Indeed, J. T. Payte et al, Curr. Ther. Res. 13, 412-416 (1971), report that acid hydrolysis of human urine samples increases the sensitivity for detecting morphine by both thin layer and gas-liquid chromatography.
Hydrolysis of the glucuronide metabolite in urine can be performed either with acid or with the enzyme .beta.-glucuronidase. When hydrolysis is used for laboratory analysis it usually is acid hydrolysis, acids being less costly than enzyme preparations and requiring much less time. F. Fish et al, J. Forens. Sci. 19, 676-683 (1974), made a detailed study of various conditions for both acid and enzymatic hydrolysis of morphine glucuronide. Those authors found a wide variation in the potency of .beta.-glucuronidase, depending on the source. Overnight incubation of addicts' pooled urine samples at 37.degree. C. using .beta.-glucuronidase from Escherichia coli, Helix pomatia, Patella vulgata and bovine liver resulted in Escherichia coli .beta.-glucuronidase consistently exhibiting greatest efficiency (64% of available morphine being liberated under optimal conditions of pH and concentration). Studies of the E. coli preparation also were conducted at 60.degree. C. in an effort to reduce the lengthy incubation time, but failed to liberate more than 5% of the available morphine. The authors concluded that the yield obtained from enzyme hydrolysis was disappointing compared to that for acid hydrolysis (which gave 93% yield after 30 minutes); enzyme hydrolysis also required more stringent control of reaction conditions. Moreover, Escherichia coli .beta.-glucuronidase, e.g. Sigma Type II employed by Fish et al, is prohibitively expensive for routine laboratory analysis.
There appear to be two reports of enzyme hydrolysis attempted at temperatures higher than about 37.degree. C. which predate Fish et al's work. Cox, Biochem. J. 71, 763-768 (1959) reported work done with .beta.-glucuronidase prepared from the visceral humps of the limpet Cellana tramoserica. Cox studied the interaction of factors including pH, time, enzyme concentration and temperature on the hydrolysis of phenolphthalein glucuronide, preparatory to a study of the optimum conditions for hydrolysis of enzyme conjugates, and among other findings noted an increase in reaction velocity up to about 60.degree. C., followed by rapid heat-denaturation of the enzyme. Experiments were conducted at 57.degree. C. and 37.degree. C., for 1 hour and 2 hour periods, at enzyme concentrations of 0.1 mg/mL and 0.05 mg/mL, at pH 3.65 to 5.25. Cox apparently did not recognize the possible significance of his elevated temperature work; there do not appear to be reports of his extending his work to hydrolysis of glucuronides other than phenolphthalein glucuronide, or to other enzyme sources. The enzyme source he employed is not commercially available.
Vela et al, Clin. Chem. 14, 837-838 (1968) reported one-hour enzymatic hydrolysis using Ketodase (a bovine liver .beta.-glucuronidase product of Sigma) applied to the gas-liquid chromatographic analysis of urinary pregnanolone, pregnanediol and pregnanetriol. The authors indicated that an increase in concentration of Ketodase to 4000 U/ml of urine at pH 4.5 with a 1 hour incubation at 60.degree. C. gave comparable results to those obtained with a 24 hour hydrolysis using 300 U/ml of urine at 37.degree. C. However, Fish et al, as discussed above, in working with morphine glucuronide, were unable to improve results by raising the temperature; indeed, the lengthy incubation time could not be shortened and the results obtained were considerably worse at 60.degree. C. than at 37.degree. C.
The snail, Helix pomatia, has been examined as a source of .beta.-glucuronidase by a number of investigators. Shackleton et al, Clin. Chim. Acta 21, 105-118 (1968) have reported use of crop fluid from Helix pomatia for enzymatic hydrolysis as part of a technique for obtaining a urinary neutral steroid profile analysis in adults and infants. Urine samples were adjusted to pH 5, 0.1 mL of crop fluid was added per 10 mL urine, the mixture was incubated at 37.degree. for 24 hours, an equal quantity of enzyme was added and incubation was continued for a further 24 hours. The authors suggest that enzymatic hydrolysis may be speeded up by increasing the enzyme concentration.
Houghton et al, Xenobiotica 9, 269-279, used Helix pomatia juice in studies related to the metabolism of anabolic steroids in the horse. Enzyme hydrolysis involved incubating 50 mL urine at pH 5 with 0.25 mL Helix pomatia juice for 36 hours at 37.degree. C.; the authors suggest, however, that the hydrolysis may have been incomplete.
Truhaut et al, C. R. Acad. Sc. Paris, Ser. D 275, 877-881 (1972), used Helix pomatia for enzymatic hydrolysis at 37.degree. for 24 hours; subsequent gas chromatography, both before and after silylation, was then used to detect heroin and its metabolites (e.g. morphine) in the urine of drug addicts.
Predmore et al, J. Forensic Sci. 19(3), 481-489 (1978) used acid hydrolysis and enzymatic hydrolysis with Helix pomatia obtained from Calbiochem for the recovery of morphine from dog urine. The authors suggest that it may take considerably longer than 16 hours to enzymatically obtain results comparable to acid hydrolysis (about 80% recovery obtained after 40 hours).
A number of investigators have studied the properties of .beta.-glucuronidase obtained from the limpet, Patella vulgata. This marine mollusc has been recognized for a number of years as a rich source of both arylsulfatase and .beta.-glucuronidase. Levvy et al, Biochem. J. 65, 203-208 (1957), found the visceral hump of this limpet to be exceptionally high in .beta.-glucuronidase activity in comparison with most animal tissues, and indicated that Patella vulgata may be one of the best sources of this enzyme for use in the hydrolysis of steroid glucuronides in urine. The authors worked at 0.degree. and 37.degree. C. at various pH's and found the optimum conditions for enzymatic hydrolysis of phenolphthalein glucuronide to be 1 hour at 37.degree. C. and pH 3.8. See also Glucuronic Acid, Free and Combined, ed. G. J. Dutton, Academic Press, New York, N.Y., 1966, 301-357; Fishman,Adv. Enzymol., 361-388 (1955); Stitch et al, Nature 172, 398-399 (1953); Dodgson et al, Biochem J. 55, 253-259 (1953). Dodgson et al, who studied a variety of marine molluscs, reported an optimum pH of 4.0 for P. vulgata in p-chlorophenylglucuronide substrate, and found that activity of the enzyme preparations increased 7 or 8 times by raising the incubation temperature from 10.degree. to 37.5.degree. C.
Wakabayashi et al, J. Biol. Chem. 236, 996-1001 (1961) studied the comparative ability of various .beta.-glucuronidase preparations, namely beef liver, Escherichia coli, Helix pomatia and Patella vulgata, to hydrolyze certain steroid glucosiduronic acids. The hydrolyses were conducted at 37.degree. C. for 1 hour at various pH levels; then, the time course of hydrolysis for each enzyme preparation and substrate was measured at its optimal pH. No one enzyme preparation was found to be uniquely superior with regard to efficiency of hydrolysis of all substrates studied. Compare Fish et al's more recent comparison of .beta.-glucuronidase from the same four sources in hydrolyzing morphine glucuronide discussed hereinabove.
Not a great deal of work has been done in the past which has utilized hydrolysis of body fluids other than urine in attempting to improve the detection of drugs therein. Berkowitz et al, Clin. Pharm. and Ther. 17, 629-635 (1975), have reported acid hydrolysis of whole human serum in an autoclave; conjugated morphine levels were found to be below free morphine levels following intravenous administration until 2 hours post dosing, with a maximum ratio of conjugated to free drug of 3:1. Acid hydrolysis at such elevated temperatures, however, denatures serum proteins, which could trap some of the morphine; such hydrolysis itself may also destroy some of the morphine.
Sensitive and reliable detection, and often quantitation, of morphine and its pharmacologically active analogues are important in many forensic, medical and other laboratory situations, such as in the diagnosis of narcotic abuse (e.g. in connection with parole violation), in investigations into cause of death and in the detection of illegal administration of such compounds in race horses or dogs. Indeed, because the narcotic analgesics and related compounds are often used in equine medicine to control pain and occasionally for their central stimulant actions, the use of these drugs in performance horses is usually prohibited. Unfortunately, the detection and quantitation of morphine and its analogues in equine body fluids, especially urine, are complicated by several factors. In the first place, the dose of drug given to a horse may be relatively small (e.g. 0.1 mg/kg or smaller in the case of morphine). Secondly, equine urine contains large amounts of mucus (from goblet cells in the epithelium and compound tubular glands in the mucous coat of the equine renal pelvis) and glucuronide derivatives of other compounds (e.g. steroids) which further interfere with the recovery of a drug such as morphine or its glucuronide metabolites from horse urine. In fact, urine from a horse is among the most difficult biological fluids in which to reduce contaminants adequately when attempting very sensitive assay methods.
As indicated above, most morphine is excreted bound as the glucuronide. Uncoupling of the morphine from its glucuronide by hydrolysis can, therefore, markedly increase the concentration of detectable morphine, which may be crucial when screening for the low concentration of morphine which may be administered to a horse. (The same would hold true for screening for other narcotics whose metabolism includes conjugation with glucuronic acid.) Moreover, since acid hydrolysis generally increases nonspecific contaminants in specimens [Frey et al, Clin Chim. Acta 51, 183-190 (1974)], it would appear that cleavage from the glucuronide would be best achieved enzymatically.